Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma

Abstract

Background: Glioblastoma (GBM) is a poorly immunogenic neoplasm treated with focused radiation. Immunotherapy has demonstrated synergistic survival effects with stereotactic radiosurgery (SRS) in murine GBM. GITR is a co-stimulatory molecule expressed constitutively on regulatory T-cells and by effector T-cells upon activation. We tested the hypothesis that anti-GITR monoclonal antibody (mAb) and SRS together would confer an immune-mediated survival benefit in glioma using the orthotopic GL261 glioma model.

Methods: Mice received SRS and anti-GITR 10 days after implantation. The anti-GITR mAbs tested were formatted as mouse IgG1 D265A (anti-GITR (1)) and IgG2a (anti-GITR (2a)) isotypes. Mice were randomized to four treatment groups: (1) control; (2) SRS; (3) anti-GITR; (4) anti-GITR/SRS. SRS was delivered to the tumor in one fraction, and mice were treated with mAb thrice. Mice were euthanized on day 21 to analyze the immunologic profile of tumor, spleen, and tumor draining lymph nodes.

Results: Anti-GITR (1)/SRS significantly improved survival over either treatment alone (p < .0001) with a cure rate of 24 % versus 0 % in a T-lymphocyte-dependent manner. There was elevated intratumoral CD4+ effector cell infiltration relative to Treg infiltration in mice treated with anti-GITR (1)/SRS, as well as significantly elevated IFNγ and IL-2 production by CD4+ T-cells and elevated IFNγ and TNFα production by CD8+ T-cells. There was increased mRNA expression of M1 markers and decreased expression of M2 markers in tumor infiltrating mononuclear cells. The anti-GITR (2a)/SRS combination did not improve survival, induce tumor regression, or result in Treg depletion.

Conclusions: These findings provide preclinical evidence for the use of anti-GITR (1) non-depleting antibodies in combination with SRS in GBM.

Keywords: Antibody; GITR; Gioblastoma; Immune checkpoint; Immunotherapy; Radiation.

Fig. 1

Eradication of intracranial GL261 tumors with anti-GITR (1) mAb plus SRS combination therapy. C57/BL6 mice were intracranially inoculated with 1.3 × 10⁵ GL261-luc cells, and after tumor establishment was confirmed by bioluminescence, mice were randomized into four groups of 10 mice per arm on day 7. Mice were administered focal radiation of 10 Gy 10 days after tumor implantation and/or received 200 μl anti-GITR (1) (10 mg/kg) by i.p. injection on days 10, 13, and 16 a. Mice were followed for survival b; curve-adjacent asterisks compare indicated curve to control. Tumor size was followed with bioluminescent imaging c; four representative mice are shown. Mice were sacrificed on day 21, and tumor infiltrating CD4 and CD8 T cells (gated on CD3+ cells) and Tregs (gated on CD4+ cells) were isolated and analyzed by flow cytometry d and e. Symbol and horizontal bar (e) denote single mouse and average value, respectively. *P < .05, **P < .01, ****P < .0001

Fig. 2

Survival benefit conferred by anti-GITR (1)/SRS treatment of murine glioma requires CD4+ T cells. C57/BL6 mice were inoculated with intracranial GL261-luc tumor, randomized to ≥7 mice per group and administered anti-GITR (1) and SRS as in Fig. 1. Mice were injected i.p. with 200 μl anti-CD4 (10 mg/kg) a and anti-CD8 (10 mg/kg) b depleting antibodies on days 5–7, 14, and 28 and followed for survival. Curve-adjacent asterisks compare indicated curve to control. The same control, anti-GITR (1), and anti-GITR (1)/SRS groups were used in (a) and (b) as the experiments were performed concurrently. The difference in survival between control mice and mice depleted of CD8+ T cells was statistically significant, but was not significant in the absence of CD4+ cells. FoxP3^DTR mice were inoculated intracranially with 1.3 × 10⁵ GL261-luc cells, randomized, injected i.p. with 200 μl of diphtheria toxin (50 ng/g) on day 8, 9 and every 2 days thereafter in order to achieve FoxP3+ cell depletion, administered anti-GITR (1) or SRS as in Fig. 1., and followed for tumor growth by bioluminescent imaging c. Tumor size was quantified by bioluminescence d. *P < .05; **P < .01. NS, non-significant

Fig. 3

Tumor infiltrating lymphocytes (TIL) in the anti-GITR (1)/SRS group have a Th1 immunophenotype and elevated effector to Treg ratio. C57BL/6 mice were inoculated with GL261-luc tumor, randomized to groups of ≥5, and dosed with anti-GITR (1) and SRS as in Fig. 1. Mice were sacrificed on day 21, tumor infiltrating lymphocytes were isolated, cells were stimulated with PMA/Ionomycin, fixed, and permeabilized for staining of intracellular markers. a CD8+ and b CD4+ cell populations were analyzed by flow cytometry for expression of IFNγ, TNFα, and IL-2. Intratumoral effector CD8+ and CD4+ to Treg ratios in the anti-GITR (1)/SRS group were calculated c. Symbol and horizontal bar denote single mouse and average value, respectively. *P < .05

Fig. 4

Anti-GITR (1)/SRS yields intratumoral myeloid cells with lower expression of M2 and higher expression of M1 markers. C57BL/6 mice were inoculated with GL261-luc tumor, randomized to groups of ≥5, and dosed with anti-GITR (1) and SRS as in Fig. 1. Mice were sacrificed on day 21, tumor infiltrating mononuclear cells were isolated, CD11b + CD45+ cells were sorted, and total RNA was isolated. Gene expression was calculated using real-time quantitative PCR analysis with 18 s as the endogenous control. Column dot plots illustrate gene expression of a cytokines, b cell surface molecules, c cell surface receptors, and d cellular enzymes in each treatment group. *P < .05, **P < .01, ***P < .001, ****P < .0001

Fig. 5

Lack of intracranial tumor eradication and Treg depletion with the anti-GITR IgG 2a antibody/SRS combination. C57BL/6 mice were intracranially inoculated with GL261-luc tumor, randomized to groups of ≥8, and dosed with 200 μl of anti-GITR (2a) (10 mg/kg) on day 10, 13, 16 and/or SRS (10 Gy) on day 10. Mice were followed for survival, a and tumor growth was assessed by bioluminescent imaging b. Mice were sacrificed on day 21, tumor infiltrating Tregs were isolated and analyzed by flow cytometry c. CD11b + CD45+ tumor resident microglia and tumor infiltrating mononuclear cells were isolated on day 21, analyzed by flow cytometry (gated on CD3+ cells), and mean fluorescence intensity (MFI) of FcγRIII and IV expression was calculated d. Flank tumors were established in C57BL/6 mice by subcutaneous inoculation of 2 × 10⁶ GL261-luc cells in a volume of 100 μl, and intracranial tumors were established as in Fig. 1. Mice were dosed i.p with 200 μl of anti-GITR (2a) (10 mg/kg) on days 10, 13, and 16, sacrificed on day 17, and tumor infiltrating lymphocytes were harvested and analyzed by flow cytometry for FoxP3 expression (gated on CD3+ cells) e-f. Symbol and horizontal bar denote single mouse and average value, respectively. *P < .05, ***P < .001. NS, non-significant